Structure and Function of a Pentameric TRPV3 Channel

Abstract Transient receptor potential (TRP) channels are a large, eukaryotic ion channel superfamily that control diverse physiological functions, and are therefore attractive drug targets. To date, more than 210 structures from over 20 different TRP channels have been determined, all are tetramers. Despite the wealth of structural information

there are many open questions, including the pore-dilation phenomenon, whereby prolonged activation leads to an increase in conductance, permeability to large ions, and loss of rectification. Using HS-AFM, we have discovered a hitherto unobserved pentameric state for TRPV3 that is in in a dynamic equilibrium with the

tetrameric state through membrane diffusive exchange of protomers. Simple geometric considerations to estimate the pore size, as well as the similar timescale of seconds-to-minutes we observed necessary to achieve both the pentameric state and pore-dilation currents, suggest that the pentameric state may be the structural

correlate to the so far elusive pore-dilation phenomenon. In this project we will therefore aim to (1) correlate the pentameric TRPV3 state with pore-dilation. (2) Determine an atomic structure of the pentameric state, and (3) investigate gain-of-function TRPV3 disease mutations which we hypothesize have increased occurrence of the

pentameric state. To complete these aims we will: (1) Use HS-AFM and NanoDSF to assess whether there is an increase in the population of pentamers, and a decrease in the stability the tetramers, following the addition of different well-known pore-dilation agents (diphenylboronic anhydride (DPBA) and heat (45°C)), and other

pore-dilation agents that we will discover. (2) Use molecular dynamics simulations (MDS) for equilibration of an initial pentameric model and determine the experimental pentameric structure using cryo-EM to achieve a high- resolution understanding of the pore structure and protomer interfaces in the pentamer. (3) Study two gain-of-

function mutations (M572I and Q580P) associated with the Olmsted syndrome, located at the TRPV3 protomer interface, and use HS-AFM, cryo-EM and electrophysiology measurements to assess whether these mutations increase the likelihood for the pentameric and pore-dilated state. Completion of these aims will allow us to assess

whether the increased conductance, permeability to larger ions, and loss of rectification associated with pore dilation are indeed related to a change in the oligomeric state of TRP channels, and thus provide a correlate to the yet structurally undetermined phenomenon of pore-dilation. Additionally, this work will provide a structural

explanation and mechanism for the increased channel activity following different TRPV3-related mutations and will thus further the development of drugs and therapeutic tools for treatment of TRP-channel related diseases. The results from this project are expected to provide answers to some long-standing and unresolved questions

in the field of TRP-channel research, and to pave the way for new avenues in membrane protein research in general, considering membrane diffusive protomer exchange as a process for conformation variability.